Approach 1. A region of the zebrafish embryos yolk, the yolk sycytial layer (YSL) contains essential signals for mesoderm and endoderm induction. We identified and characterized a critical YSL transcription factor, Mxtx2 over the last two years as published in Development this year. Subsequent to these studies, we have generated microarray data and Chromatin immunoprecipitation sequence data that will enable us to define the genes activated by Mxtx2 and the precise binding sites that mediate this activation. We have also defined a core set of signaling ligands that are responsible for the induction of posterior mesoderm identities downstream of Mxtx2. Approach 2. Modeling complex genetics underlying human disease in zebrafish. We have established a new zebrafish mutant genetic line in a Nodal pathway antagonist and have used this line in a series of studies to establish that simultaneously carrying Nodal and Nodal antagonist mutations confers protection against the acquistion of cyclopia, analogous to human holoprosencephaly, but increases the risk of laterality defects. Approach 3. Identifying roles of RhoGTPase signaling in gastrulation movements. Background: RhoGEF proteins are positive regulators of RhoGTPases, which have profound roles in cellular movement and morphology. To identify RhoGEFs with roles in directing the morphogenetic events of gastrulation, we performed a loss-of-function screen. We identified 48 RhoGEFs expressed during early embryogenesis and determined the loss-of-function phenotypes for 23 of these, using a non-invasive embryo holding system we designed that allows for the parallel time-lapse documention of 54 embryos. We thus identified five RhoGEFs for which two independent MOs produced the same phenotype. Three of these, homologues of ARHGEF16, Frabin and Net1, respectively, disrupted epiboly. Two others, ARHGEF10 and PLEKHG4 homologues, caused post-gastrulation defects during somitogenesis stages. We have demonstrated the specificity of the Frabin and Net1 MOs by performing mRNA rescue experiments. A manuscript describing this work is being prepared. Approach 4. Defining roles and interactions of transcription factors in the early mesoderm and endoderm. We previously developed a novel approach to identify many of the genes expressed in nascent mesoderm and endoderm. This year we have embarked in a revisitation of this project, but using deep sequencing to get a much more exhaustive list of expressed genes. Our analysis of initial deep sequence data is ongoing.